Fluorescent dyes have many uses and are known to be particularly suitable for biological applications in which the high detectability of fluorescence is desirable. By binding to a specific biological ingredient in a sample, a fluorescent dye can be used to indicate the presence or the quantity of the specific ingredient in a sample. A variety of fluorescent dyes are available for specific fluorescent staining and quantitation of DNA and RNA, and other applications involving nucleic acids.
Unsymmetrical cyanine dyes were described long before much was known about DNA by Brooker, et al., J. AM. CHEM. SOC. 64, 199 (1942). These dyes have since been found to be useful in fluorescent staining of DNA and RNA. The dye sold under the tradename Thiazole Orange has particular advantages in the quantitative analysis of immature blood cells or reticulocytes. U.S. Pat. No. 4,883,867 to Lee, et al. (1989) ('867 patent); Lee, et al., Thiazole Orange: A New Dye for Reticulocyte Analysis, CYTOMETRY 7, 508 (1986). As indicated in the '867 patent to Lee, et al., the dye used for this purpose must be able to penetrate the cell membrane.
The inventors have discovered that a composition that includes two suitably connected unsymmetrical cyanine dye units, i.e. a cyanine dye dimer, is a polar compound that is unable to readily penetrate cell membranes. Nevertheless, the composition discovered by inventors is highly useful as a stain for nucleic acids because it is sensitive to even small fragments of nucleic acid polymers not contained inside living cells, e.g. in cell extracts, as well as to nucleic acids in permeabilized cells. The dimer is neither anticipated nor obvious in view of Thiazole Orange or related compounds that are monomers.
Other dimer compounds that are known to bind to nucleic acids with a large fluorescence enhancement include variants of ethidium homodimer, acridine homodimers, acridine-ethidium heterodimer, and 7-hydropyridocarbazoles, see, e.g., Rye, et al., NUCLEIC ACIDS RESEARCH 19(2), 327 (1990); Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS Set 28 (1989). Although the Rye, et al. reference mentions characteristics that influence the affinity and mode of binding dimers to DNA, the reference does not describe the compounds used in this invention. The novel dimer compounds described herein are not only different in structure from other dimer compounds but are also superior to other dimers and to Thiazole Orange in their sensitivity to nucleic acids.